Treatment of Pompe&#39;s Disease

ABSTRACT

The invention provides methods of treating Pompe&#39;s disease using human acid alpha glucosidase. A preferred treatment regime comprises administering greater than 10 mg/kg body weight per week to a patient.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/012,003, filed Jan. 30, 2008 which is a continuation of U.S.application Ser. No. 10/611,598, filed Jun. 30, 2003 which is acontinuation of U.S. application Ser. No. 09/454,711, filed Dec. 6,1999, which claims the benefit of U.S. Provisional Application No.60/111,291 filed Dec. 7, 1998, which is related to U.S. application Ser.No. 08/700,760 filed Jul. 29, 1996, now U.S. Pat. No. 6,118,045, whichclaims the benefit of U.S. Provisional Application No. 60/001,796, filedAug. 2, 1995. The entire teachings of the above applications areincorporated herein by reference.

TECHNICAL FIELD

The present invention resides in the fields of recombinant genetics, andmedicine, and is directed to enzyme-replacement therapy of patients withPompe's disease.

BACKGROUND OF THE INVENTION

Like other secretory proteins, lysosomal proteins are synthesized in theendoplasmic reticulum and transported to the Golgi apparatus. However,unlike most other secretory proteins, the lysosomal proteins are notdestined for secretion into extracellular fluids but into anintracellular organelle. Within the Golgi, lysosomal proteins undergospecial processing to equip them to reach their intracellulardestination. Almost all lysosomal proteins undergo a variety ofposttranslational modifications, including glycosylation andphosphorylation via the 6′ position of a terminal mannose group. Thephosphorylated mannose residues are recognized by specific receptors onthe inner surface of the Trans Golgi Network. The lysosomal proteinsbind via these receptors, and are thereby separated from other secretoryproteins. Subsequently, small transport vesicles containing thereceptor-bound proteins are pinched off from the Trans Golgi Network andare targeted to their intracellular destination. See generally Kornfeld,Biochem. Soc. Trans. 18, 367-374 (1990).

There are over thirty lysosomal diseases, each resulting from adeficiency of a particular lysosomal protein, usually as a result ofgenetic mutation. See, e.g., Cotran et al., Robbins Pathologic Basis ofDisease (4th ed. 1989) (incorporated by reference in its entirety forall purposes). The deficiency in the lysosomal protein usually resultsin harmful accumulation of a metabolite. For example, in Hurler's,Hunter's, Morquio's, and Sanfilippo's syndromes, there is anaccumulation of mucopolysaccharides; in Tay-Sachs, Gaucher, Krabbe,Niemann-Pick, and Fabry syndromes, there is an accumulation ofsphingolipids; and in fucosidosis and mannosidosis, there is anaccumulation of fucose-containing sphingolipids and glycoproteinfragments, and of mannose-containing oligosaccharides, respectively.

Glycogen storage disease type II (GSD II; Pompe disease; acid maltasedeficiency) is caused by deficiency of the lysosomal enzyme acidα-glucosidase (acid maltase). Two clinical forms are distinguished:early onset infantile and late onset, juvenile and adult. Infantile GSDII has its onset shortly after birth and presents with progressivemuscular weakness and cardiac failure. This clinical variant is usuallyfatal within the first two years of life. Symptoms in the late onset inadult and juvenile patients occur later in life, and only skeletalmuscles are involved. The patients eventually die due to respiratoryinsufficiency. Patients may exceptionally survive for more than sixdecades. There is a good correlation between the severity of the diseaseand the residual acid α-glucosidase activity, the activity being 10-20%of normal in late onset and less than 2% in early onset forms of thedisease (see Hirschhorn, The Metabolic and Molecular Bases of InheritedDisease (Scriver et al., eds., 7th ed., McGraw-Hill, 1995), pp.2443-2464).

Since the discovery of lysosomal enzyme deficiencies as the primarycause of lysosomal storage diseases (see, e.g., Hers, Biochem. J. 86,11-16 (1963)), attempts have been made to treat patients havinglysosomal storage diseases by (intravenous) administration of themissing enzyme, i.e., enzyme therapy. These experiments with enzymereplacement therapy for Pompe's disease were not successful. Eithernon-human α-glucosidase from Aspergillus niger, giving immunologicalreactions, or a form of the enzyme that is not efficiently taken up bycells (the low uptake form, mature enzyme from human placenta; seebelow) was used. Moreover, both the duration of treatment, and/or theamount of enzyme administered were insufficient (3-5). Production oflysosomal enzymes from natural sources such as human urine and bovinetestis is in theory possible, but gives low yields, and the enzymepurified is not necessarily in a form that can be taken up by tissues ofa recipient patient.

Notwithstanding the above uncertainties and difficulties, the inventionprovides methods of treating patients for Pompe's disease using humanacid alpha glucosidase.

SUMMARY OF THE INVENTION

In one aspect, the invention provides methods of treating a patient withPompe's disease. Such methods entail administering to the patient atherapeutically effective amount of human acid alpha glucosidase. Thedosage is preferably at least 10 mg/kg body weight per week. In somemethods, the dosage is at least 60 mg/kg body weight per week or atleast 120 mg/kg body weight per week. In some methods, such dosages areadministered on a single occasion per week and in other methods on threeoccasions per week. In some methods, the treatment is continued for ateleast 24 weeks. Administration is preferably intravenous. The human acidalpha glucosidase is preferably obtained in the milk of a nonhumantransgenic mammal, and is preferably predominantly in a 110 kD form.

The methods can be used for treating patients with infantile, juvenileor adult Pompe's disease. In some methods of treating infantile Pompe'sdisease efficacy is indicated by a patient surviving to be at least oneyear old.

In some methods, levels of human acid alpha glucosidase are monitored inthe patient. Optionally, a second dosage of human acid alpha glucosidasecan be administered if the level of alpha-glucosidase falls below athreshold value in the patient.

In some methods, the human alpha glucosidase is administeredintravenously and the rate of administration increases during the periodof administration. In some methods, the rate of administration increasesby at least a factor of ten during the period of administration. In somemethods, the rate of administration increases by at least a factor often within a period of five hours. In some methods, the patient isadministered a series of at least four dosages, each dosage at a higherstrength than the previous dosage. In some methods, the dosages are afirst dosage of 0.03-3 mg/kg/hr, a second dosage of 0.3-12 mg/kg/hr, athird dosage of 1-30 mg/kg/hr and a fourth dosage of 2-60 mg/kg/hr. Insome methods, the dosages are a first dosage of 0.1-1 mg/kg/hr, a seconddosage of 14 mg/kg/hr, a third dosage of 3-10 mg/kg/hr and a fourthdosage of 6-20 mg/kg/hr. In some methods, the dosages are a first dosageof 0.254 mg/kg/hr, a second dosage of 0.9-1.4 mg/kg/hr, a third dosageof 3.6-5.7 mg/kg/hr and a fourth dosage of 7.211.3 mg/kg/hr. In somemethods, the dosages are a first dosage of 0.3 mg/kg/hr, a second dosageof 1 mg/kg/hr, a third dosage of 4 mg/kg/hr and a fourth dosage of 12mg/kg/hr. In some methods, the first, second, third and fourth dosagesare each administered for periods of 15 min to 8 hours.

In some methods, the first, second, third and fourth dosages areadministered for periods of 1 hr, 1 hr, 0.5 hr and 3 hr respectively.

In another aspect, the invention provides a pharmaceutical compositioncomprising human acid alpha glucosidase, human serum albumin, and asugar in a physiologically acceptable buffer in sterile form. Some suchcompositions comprise human acid alpha glucosidase, human serum albumin,and glucose in sodium phosphate buffer. Some compositions comprise alphaglucosidase, mannitol and sucrose in an aqueous solution. In somecompositions, the sugar comprises mannitol and sucrose and theconcentration of mannitol is 1-3% w/w of the aqueous solution and theconcentration of sucrose is 0.1 to 1% w/w of the aqueous solution. Insome compositions, the concentration of mannitol is 2% w/w and theconcentration of sucrose is 0.5% w/w.

The invention further provides a lyophilized composition produced bylyophilizing a pharmaceutical composition comprising human acidglucosidase, mannitol and sucrose in aqueous solution. Such acomposition can be prepared by lyophilizing a first compositioncomprising human acid alpha-glucosidase, mannitol, sucrose and anaqueous solution to produce a second composition; and reconstituting thelyophilized composition in saline to produce a third composition. Insome such compositions, the human acid alpha-glucosidase is at 5 mg/mlin both the first and third composition, the mannitol is at 2 mg/ml inthe first composition, the sucrose is at 0.5 mg/ml in the firstcomposition, and the saline used in the reconstituting step is 0.9% w/w.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A transgene containing acid α-glucosidase cDNA. The αs1-caseinexons are represented by open boxes; α-glucosidase cDNA is representedby a shaded box. The αs1-casein intron and flanking sequences (SEQ IDNOS:2 and 3) are represented by a thick line. A thin line represents theIgG acceptor site. The transcription initiation site is marked (1^(→)),the translation initiation site (ATG), the stop codon (TAG) and thepolyadenylation site (pA).

FIG. 2 (panels A, B, C): Three transgenes containing acid α-glucosidasegenomic DNA. Dark shaded areas are αs1 casein sequences, open boxesrepresent acids α-glucosidase exons, and the thin line between the openboxes represents α-glucosidase introns. Other symbols are the same as inFIG. 1

FIG. 3 (panels A, B, C): Construction of genomic transgenes. Theα-glucosidase exons are represented by open boxes; the α-glucosidaseintrons and nontranslated sequences are indicated by thin lines. ThepKUN vector sequences are represented by thick lines.

FIGS. 4A and 4B. Detection of acid α-glucosidase in milk of transgenicmice by Western blotting.

DEFINITIONS

The term “substantial identity” or “substantial homology” means that twopeptide sequences, when optimally aligned, such as by the programs GAPor BESTFIT using default gap weights, share at least 65 percent sequenceidentity, preferably at least 80 or 90 percent sequence identity, morepreferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions.

The term “substantially pure” or “isolated” means an object species hasbeen identified and separated and/or recovered from a component of itsnatural environment. Usually, the object species is the predominantspecies present (i.e., on a molar basis it is more abundant than anyother individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 to 90 percent by weight ofall macromolecular species present in the composition. Most preferably,the object species is purified to essential homogeneity (contaminantspecies cannot be detected in the composition by conventional detectionmethods) wherein the composition consists essentially of derivatives ofa single macromolecular species.

A DNA segment is operably linked when placed into a functionalrelationship with another DNA segment. For example, DNA for a signalsequence is operably linked to DNA encoding a polypeptide if it isexpressed as a preprotein that participates in the secretion of thepolypeptide; a promoter or enhancer is operably linked to a codingsequence if it stimulates the transcription of the sequence. Generally,DNA sequences that are operably linked are contiguous, and in the caseof a signal sequence both contiguous and in reading phase. However,enhancers need not be contiguous with the coding sequences whosetranscription they control. Linking is accomplished by ligation atconvenient restriction sites or at adapters or linkers inserted in lieuthereof.

An exogenous DNA segment is one foreign to the cell, or homologous to aDNA segment of the cell but in an unnatural position in the host cellgenome. Exogenous DNA segments are expressed to yield exogenouspolypeptides.

In a transgenic mammal, all, or substantially all, of the germline andsomatic cells contain a transgene introduced into the mammal or anancestor of the mammal at an early embryonic stage.

DETAILED DESCRIPTION

The invention provides transgenic nonhuman mammals secreting a lysosomalprotein into their milk. Secretion is achieved by incorporation of atransgene encoding a lysosomal protein and regulatory sequences capableof targeting expression of the gene to the mammary gland. The transgeneis expressed, and the expression product posttranslationally modifiedwithin the mammary gland, and then secreted in milk. Theposttranslational modification can include steps of glycosylation andphosphorylation to produce a mannose-6 phosphate containing lysosomalprotein.

A. Lysosomal Genes

The invention provides transgenic nonhuman mammals expressing DNAsegments containing any of the more than 30 known genes encodinglysosomal enzymes and other types of lysosomal proteins, includingα-glucosidase, α-L-iduronidase, iduronate-sulfate sulfatase,hexosaminidase A and B, ganglioside activator protein, arylsulfatase Aand B, iduronate sulfatase, heparan N-sulfatase, galacto-ceramidase,α-galactosylceramidase A, sphingomyelinase, α-fucosidase, α-mannosidase,aspartylglycosamine amide hydrolase, acid lipase,N-acetyl-a-D-glucosamine-6-sulphate sulfatase, α- and β-galactosidase,β-glucuronidase, β-mannosidase, ceramidase, galacto-cerebrosidase,α-N-acetylgalactosaminidase, and protective protein and others.Transgenic mammals expressing allelic, cognate and induced variants ofany of the known lysosomal protein gene sequences are also included.Such variants usually show substantial sequence identity at the aminoacid level with known lysosomal protein genes. Such variants usuallyhybridize to a known gene under stringent conditions or crossreact withantibodies to a polypeptide encoded by one of the known genes.

DNA clones containing the genomic or cDNA sequences of many of the knowngenes encoding lysosomal proteins are available. (Scott et al., Am. J.Hum. Genet. 47, 802-807 (1990); Wilson et al., PNAS 87, 8531-8535(1990); Stein et al., J. Biol. Chem. 264, 1252-1259 (1989); Ginns etal., Biochem. Biophys. Res. Comm. 123, 574-580 (1984); Hoefsloot et al.,EMBO J. 7, 1697-1704 (1988); Hoefsloot et al., Biochem. J. 272, 473-479(1990); Meyerowitz and Proia, PNAS 81, 5394-5398 (1984); Scriver et al.,supra, part 12, pages 2427-2882 and references cited therein)) Otherexamples of genomic and cDNA sequences are available from GenBank. Tothe extent that additional cloned sequences of lysosomal genes arerequired, they may be obtained from genomic or cDNA libraries(preferably human) using known lysosomal protein DNA sequences orantibodies to known lysosomal proteins as probes.

B. Conformation of Lysosomal Proteins

Recombinant lysosomal proteins are preferably processed to have the sameor similar structure as naturally occurring lysosomal proteins.Lysosomal proteins are glycoproteins that are synthesized on ribosomesbound to the endoplasmic reticulum (RER). They enter this organelleco-translationally guided by an N-terminal signal peptide (Ng et al.,Current Opinion in Cell Biology 6, 510-516 (1994)). The N-linkedglycosylation process starts in the RER with the en bloc transfer of thehigh-mannose oligosaccharide precursor Glc3Man9GlcNAc2 from a dolicholcarrier. Carbohydrate chain modification starts in the RER and continuein the Golgi apparatus with the removal of the three outermost glucoseresidues by glycosidases I and II. Phosphorylation is a two-stepprocedure in which first N-acetyl-gluco-samine-1-phosphate is coupled toselect mannose groups by a lysosomal protein specific transferase, andsecond, the N-acetyl-glucosamine is cleaved by a diesterase (Goldberg etal., Lysosomes: Their Role in Protein Breakdown (Academic Press Inc.,London, 1987), pp. 163-191). Cleavage exposes mannose 6-phosphate as arecognition marker and ligand for the mannose 6-phosphate receptormediating transport of most lysosomal proteins to the lysosomes(Kornfeld, Biochem. Soc. Trans. 18, 367-374 (1992)).

In addition to carbohydrate chain modification, most lysosomal proteinsundergo proteolytic processing, in which the first event is removal ofthe signal peptide. The signal peptide of most lysosomal proteins iscleaved after translocation by signal peptidase after which the proteinsbecome soluble. There is suggestive evidence that the signal peptide ofacid α-glucosidase is cleaved after the enzyme has left the RER, butbefore it has entered the lysosome or the secretory pathway (Wisselaaret al., J. Biol. Chem. 268, 2223-2231 (1993)). The proteolyticprocessing of acid at-glucosidase is complex and involves a series ofsteps in addition to cleavage of the signal peptide taking place atvarious subcellular locations. Polypeptides are cleaved off at both theN and C terminal ends, whereby the specific catalytic activity isincreased. The main species recognized are a 110/100 kD precursor, a 95kD intermediate and 76 kD and 70 kD mature forms. (Hasilik et al., J.Biol. Chem. 255, 4937-4945 (1980); Oude Elferink et al., Eur. J.Biochem. 139, 489-495 (1984); Reuser et al., J. Biol. Chem. 260,8336-8341 (1985); Hoefsloot et al., EMBO J. 7, 1697-1704 (1988)). Thepost translational processing of natural human acid α-glucosidase and ofrecombinant forms of human acid α-glucosidase as expressed in culturedmammalian cells like COS cells, BHK cells and CHO cells is similar(Hoefsloot et al., (1990) supra; Wisselaar et al., (1993) supra.

Authentic processing to generate lysosomal proteins phosphorylated atthe 6′ position of the mannose group can be tested by measuring uptakeof a substrate by cells bearing a receptor for mannose 6-phosphate.Correctly modified substrates are taken up faster than unmodifiedsubstrates, and in a manner whereby uptake of the modified substrate canbe competitively inhibited by addition of mannose 6-phosphate.

C. Transgene Design

Transgenes are designed to target expression of a recombinant lysosomalprotein to the mammary gland of a transgenic nonhuman mammal harboringthe transgene. The basic approach entails operably linking an exogenousDNA segment encoding the protein with a signal sequence, a promoter andan enhancer. The DNA segment can be genomic, minigene (genomic with oneor more introns omitted), cDNA, a YAC fragment, a chimera of twodifferent lysosomal protein genes, or a hybrid of any of these.Inclusion of genomic sequences generally leads to higher levels ofexpression. Very high levels of expression might overload the capacityof the mammary gland to perform posttranslation modifications, andsecretion of lysosomal proteins. However, the data presented belowindicate that substantial posttranslational modification occursincluding the formation of mannose 6-phosphate groups, notwithstanding ahigh expression level in the mg/ml range. Substantial modification meansthat at least about 10, 25, 50, 75 or 90% of secreted molecules bear atleast one mannose 6-phosphate group. Thus, genomic constructs or hybridcDNA-genomic constructs are generally preferred.

In genomic constructs, it is not necessary to retain all intronicsequences. For example, some intronic sequences can be removed to obtaina smaller transgene facilitating DNA manipulations and subsequentmicroinjection. See Archibald et al., WO 90/05188 (incorporated byreference in its entirety for all purposes). Removal of some introns isalso useful in some instances to reduce expression levels and therebyensure that posttranslational modification is substantially complete. Inother instances excluding an intron such as intron one from the genomicsequence of acid α-glucosidase leads to a higher expression of themature enzyme. It is also possible to delete some or all of noncodingexons. In some transgenes, selected nucleotides in lysosomal proteinencoding sequences are mutated to remove proteolytic cleavage sites.

Because the intended use of lysosomal proteins produced by transgenicmammals is usually administration to humans, the species from which theDNA segment encoding a lysosomal protein sequence is obtained ispreferably human. Analogously if the intended use were in veterinarytherapy (e.g., on a horse, dog or cat), it is preferable that the DNAsegment be from the same species.

The promoter and enhancer are from a gene that is exclusively or atleast preferentially expressed in the mammary gland (i.e., amammary-gland specific gene).

Preferred genes as a source of promoter and enhancer include β-casein,κ-casein, αS1-casein, αS2-casein, β-lactoglobulin, whey acid protein,and α-lactalbumin. The promoter and enhancer are usually but not alwaysobtained from the same mammary-gland specific gene. This gene issometimes but not necessarily from the same species of mammal as themammal into which the transgene is to be expressed. Expressionregulation sequences from other species such as those from human genescan also be used. The signal sequence must be capable of directing thesecretion of the lysosomal protein from the mammary gland. Suitablesignal sequences can be derived from mammalian genes encoding a secretedprotein. Surprisingly, the natural signal sequences of lysosomalproteins are suitable, notwithstanding that these proteins are normallynot secreted but targeted to an intracellular organelle. In addition tosuch signal sequences, preferred sources of signal sequences are thesignal sequence from the same gene as the promoter and enhancer areobtained. Optionally, additional regulatory sequences are included inthe transgene to optimize expression levels. Such sequences include 5′flanking regions, 5′ transcribed but untranslated regions, intronicsequences, 3′ transcribed but untranslated regions, polyadenylationsites, and 3′ flanking regions. Such sequences are usually obtainedeither from the mammary-gland specific gene from which the promoter andenhancer are obtained or from the lysosomal protein gene beingexpressed. Inclusion of such sequences produces a genetic milieusimulating that of an authentic mammary gland specific gene and/or thatof an authentic lysosomal protein gene. This genetic milieu results insome cases (e.g., bovine αS1-casein) in higher expression of thetranscribed gene. Alternatively, 3′ flanking regions and untranslatedregions are obtained from other heterologous genes such as the P-globingene or viral genes. The inclusion of 3′ and 5′ untranslated regionsfrom a lysosomal protein gene, or other heterologous gene can alsoincrease the stability of the transcript.

In some embodiments, about 0.5, 1, 5, 10, 15, 20 or 30 kb of 5′ flankingsequence is included from a mammary specific gene in combination withabout 1, 5, 10, 15, 20 or 30 kb or 3′ flanking sequence from thelysosomal protein gene being expressed. If the protein is expressed froma cDNA sequence, it is advantageous to include an intronic sequencebetween the promoter and the coding sequence. The intronic sequence ispreferably a hybrid sequence formed from a 5′ portion from anintervening sequence from the first intron of the mammary gland specificregion from which the promoter is obtained and a 3′ portion from anintervening sequence of an IgG intervening sequence or lysosomal proteingene. See DeBoer et al., WO 91/08216 (incorporated by reference in itsentirety for all purposes).

A preferred transgene for expressing a lysosomal protein comprises acDNA-genomic hybrid lysosomal protein gene-linked 5′ to a caseinpromoter and enhancer. The hybrid gene includes the signal sequence,coding region, and a 3′ flanking region from the lysosomal protein gene.Optionally, the cDNA segment includes an intronic sequence between the5′ casein and untranslated region of the gene encoding the lysosomalprotein. Of course, corresponding cDNA and genomic segments can also befused at other locations within the gene provided a contiguous proteincan be expressed from the resulting fusion.

Other preferred transgenes have a genomic lysosomal protein segmentlinked 5′ to casein regulatory sequences. The genomic segment is usuallycontiguous from the 5′ untranslated region to the 3′ flanking region ofthe gene. Thus, the genomic segment includes a portion of the lysosomalprotein 5′ untranslated sequence, the signal sequence, alternatingintrons and coding exons, a 3′ untranslated region, and a 3′ flankingregion. The genomic segment is linked via the 5′ untranslated region toa casein fragment comprising a promoter and enhancer and usually a 5′untranslated region.

DNA sequence information is available for all of the mammary glandspecific genes listed above, in at least one, and often severalorganisms. See, e.g., Richards et al., J. Biol. Chem. 256, 526-532(1981) (α-lactalbumin rat); Campbell et al., Nucleic Acids Res. 12,8685-8697 (1984) (rat WAP); Jones et al., J. Biol. Chem. 260, 7042-7050(1985)) (rat. β-casein); Yu-Lee and Rosen, J. Biol. Chem. 258,10794-10804 (1983) (rat γ casein)); Hall, Biochem. J. 242, 735-742(1987) (α-lactalbumin human); Stewart, Nucleic Acids Res. 12, 389 (1984)(bovine αs1 and K casein cDNAs); Gorodetsky et al., Gene 66, 87-96(1988) (bovine (3 casein); Alexander et al., Eur. J. Biochem. 178,395-401 (1988) (bovine κ casein); Brignon et al., FEBS Lett. 188, 48-55(1977) (bovine αS2 casein); Jamieson et al., Gene 61, 85-90 (1987),Ivanov et al., Biol. Chem. Hoppe-Seyler 369, 425-429 (1988), Alexanderet al., Nucleic Acids Res. 17, 6739 (1989) (bovine β lactoglobulin);Vilotte et al., Biochimie 69, 609-620 (1987) (bovine α-lactalbumin)(incorporated by reference in their entirety for all purposes). Thestructure and function of the various milk protein genes are reviewed byMercier and Vilotte, J. Dairy Sci. 76, 3079-3098 (1993) (incorporated byreference in its entirety for all purposes). To the extent thatadditional sequence data might be required, sequences flanking theregions already obtained could be readily cloned using the existingsequences as probes. Mammary-gland specific regulatory sequences fromdifferent organisms are likewise obtained by screening libraries fromsuch organisms using known cognate nucleotide sequences, or antibodiesto cognate proteins as probes.

General strategies and exemplary transgenes employing αS1-caseinregulatory sequences for targeting the expression of a recombinantprotein to the mammary gland are described in more detail in DeBoer etal., WO 91/08216 and WO 93/25567 (incorporated by reference in theirentirety for all purposes). Examples of transgenes employing regulatorysequences from other mammary gland specific genes have also beendescribed. See, e.g., Simon et al., Bio/Technology 6, 179-183 (1988) andWO88/00239 (1988) (β-lactoglobulin regulatory sequence for expression insheep); Rosen, EP 279,582 and Lee et al., Nucleic Acids Res. 16,1027-1041 (1988) (β-casein regulatory sequence for expression in mice);Gordon, Biotechnology 5, 1183 (1987) (WAP regulatory sequence forexpression in mice); WO 88/01648 (1988) and Eur. J. Biochem. 186, 43-48(1989) (α-lactalbumin regulatory sequence for expression in mice)(incorporated by reference in their entirety for all purposes).

The expression of lysosomal proteins in the milk from transgenes can beinfluenced by co-expression or functional inactivation (i.e., knock-out)of genes involved in post translational modification and targeting ofthe lysosomal proteins. The data in the Examples indicate thatsurprisingly mammary glands already express modifying enzymes atsufficient quantities to obtain assembly and secretion of mannose6-phosphate containing proteins at high levels. However, in sometransgenic mammals expressing these proteins at high levels, it issometimes preferable to supplement endogenous levels of processingenzymes with additional enzyme resulting from transgene expression. Suchtransgenes are constructed employing similar principles to thosediscussed above with the processing enzyme coding sequence replacing thelysosomal protein coding sequence in the transgene. It is not generallynecessary that posttranslational processing enzymes be secreted. Thus,the secretion signal sequence linked to the lysosomal protein codingsequence is replaced with a signal sequence that targets the processingenzyme to the endoplasmic reticulum without secretion. For example, thesignal sequences naturally associated with these enzymes are suitable.

D. Transgenesis

The transgenes described above are introduced into nonhuman mammals.Most nonhuman mammals, including rodents such as mice and rats, rabbits,ovines such as sheep and goats, porcines such as pigs, and bovines suchas cattle and buffalo, are suitable. Bovines offer an advantage of largeyields of milk, whereas mice offer advantages of ease of transgenesisand breeding. Rabbits offer a compromise of these advantages. A rabbitcan yield 100 ml milk per day with a protein content of about 14% (seeBuhler et al., Biotechnology 8, 140 (1990)) (incorporated by referencein its entirety for all purposes), and yet can be manipulated and bredusing the same principles and with similar facility as mice.Nonviviparous mammals such as a spiny anteater or duckbill platypus aretypically not employed.

In some methods of transgenesis, transgenes are introduced into thepronuclei of fertilized oocytes. For some animals, such as mice andrabbits, fertilization is performed in vivo and fertilized ova aresurgically removed. In other animals, particularly bovines, it ispreferable to remove ova from live or slaughterhouse animals andfertilize the ova in vitro. See DeBoer et al., WO 91/08216. In vitrofertilization permits a transgene to be introduced into substantiallysynchronous cells at an optimal phase of the cell cycle for integration(not later than S-phase). Transgenes are usually introduced bymicroinjection. See U.S. Pat. No. 4,873,292. Fertilized oocytes are thencultured in vitro until a pre-implantation embryo is obtained containingabout 16-150 cells. The 16-32 cell stage of an embryo is described as amorula. Pre-implantation embryos containing more than 32 cells aretermed blastocysts. These embryos show the development of a blastocoelecavity, typically at the 64 cell stage. Methods for culturing fertilizedoocytes to the pre-implantation stage are described by Gordon et al.,Methods Enzymol. 101, 414 (1984); Hogan et al., Manipulation of theMouse Embryo: A Laboratory Manual, C.S.H.L. N.Y. (1986) (mouse embryo);and Hammer et al., Nature 315, 680 (1985) (rabbit and porcine embryos);Gandolfi et al. J. Reprod. Fert. 81, 23-28 (1987); Rexroad et al., J.Anim. Sci. 66, 947-953 (1988) (ovine embryos) and Eyestone et al. J.Reprod. Fert. 85, 715-720 (1989); Camous et al., J. Reprod. Fert. 72,779-785 (1984); and Heyman et al. Theriogenology 27, 5968 (1987) (bovineembryos) (incorporated by reference in their entirety for all purposes).Sometimes pre-implantation embryos are stored frozen for a periodpending implantation. Pre-implantation embryos are transferred to theoviduct of a pseudopregnant female resulting in the birth of atransgenic or chimeric animal depending upon the stage of developmentwhen the transgene is integrated. Chimeric mammals can be bred to formtrue germline transgenic animals.

Alternatively, transgenes can be introduced into embryonic stem cells(ES). These cells are obtained from preimplantation embryos cultured invitro. Bradley et al., Nature 309, 255-258 (1984) (incorporated byreference in its entirety for all purposes). Transgenes can beintroduced into such cells by electroporation or microinjection.Transformed ES cells are combined with blastocysts from a non-humananimal. The ES cells colonize the embryo and in some embryos form thegermline of the resulting chimeric animal. See Jaenisch, Science, 240,1468-1474 (1988) (incorporated by reference in its entirety for allpurposes). Alternatively, ES cells can be used as a source of nuclei fortransplantation into an enucleated fertilized oocyte giving rise to atransgenic mammal.

For production of transgenic animals containing two or more transgenes,the transgenes can be introduced simultaneously using the same procedureas for a single transgene. Alternatively, the transgenes can beinitially introduced into separate animals and then combined into thesame genome by breeding the animals. Alternatively, a first transgenicanimal is produced containing one of the transgenes. A second transgeneis then introduced into fertilized ova or embryonic stem cells from thatanimal. In some embodiments, transgenes whose length would otherwiseexceed about 50 kb, are constructed as overlapping fragments. Suchoverlapping fragments are introduced into a fertilized oocyte orembryonic stem cell simultaneously and undergo homologous recombinationin vivo. See Kay et al., WO 92/03917 (incorporated by reference in itsentirety for all purposes).

E. Characteristics of Transgenic Mammals

Transgenic mammals of the invention incorporate at least one transgenein their genome as described above. The transgene targets expression ofa DNA segment encoding a lysosomal protein at least predominantly to themammary gland. Surprisingly, the mammary glands are capable ofexpressing proteins required for authentic posttranslation processingincluding steps of oligosaccharide addition and phosphorylation.Processing by enzymes in the mammary gland results in phosphorylation ofthe 6′ position of mannose groups.

Lysosomal proteins can be secreted at high levels of at least 10, 50,100, 500, 1000, 2000, 5000 or 10,000 .μg/ml. Surprisingly, thetransgenic mammals of the invention exhibit substantially normal health.Secondary expression of lysosomal proteins in tissues other than themammary gland does not occur to an extent sufficient to causedeleterious effects. Moreover, exogenous lysosomal protein produced inthe mammary gland is secreted with sufficient efficiency that nosignificant problem is presented by deposits clogging the secretoryapparatus.

The age at which transgenic mammals can begin producing milk, of course,varies with the nature of the animal. For transgenic bovines, the age isabout two-and-a-half years naturally or six months with hormonalstimulation, whereas for transgenic mice the age is about 5-6 weeks. Ofcourse, only the female members of a species are useful for producingmilk. However, transgenic males are also of value for breeding femaledescendants. The sperm from transgenic males can be stored frozen forsubsequent in vitro fertilization and generation of female offspring

F. Recovery of Proteins from Milk

Transgenic adult female mammals produce milk containing highconcentrations of exogenous lysosomal protein. The protein can bepurified from milk, if desired, by virtue of its distinguishing physicaland chemical properties, and standard purification procedures such asprecipitation, ion exchange, molecular exclusion or affinitychromatography. See generally Scopes, Protein Purification(Springer-Verlag, N.Y., 1982).

Purification of human acid α-glucosidase from milk can be carried out bydefatting of the transgenic milk by centrifugation and removal of thefat, followed by removal of caseins by high speed centrifugationfollowed by dead-end filtration (i.e., dead-end filtration by usingsuccessively declining filter sizes) or cross-flow filtration, or;removal of caseins directly by cross-flow filtration. Human acidα-glucosidase is purified by chromatography, including Q Sepharose FF(or other anion-exchange matrix), hydrophobic interaction chromatography(HIC), metal-chelating Sepharose, or lectins coupled to Sepharose (orother matrices).

Q Sepharose Fast Flow chromatography may be used to purify human acidα-glucosidase present in filtered whey or whey fraction as follows: a QSepharose Fast Flow (QFF; Pharmacia) chromatography (Pharmacia XK-50column, 15 cm bed height; 250 cm/hr flow rate) the column wasequilibrated in 20 mM sodium phosphate buffer, pH 7.0 (buffer A); theS/D-incubated whey fraction (about 500 to 600 ml) is loaded and thecolumn is washed with 4-6 column volumes (cv) of buffer A (20 mM sodiumphosphate buffer, pH 7.0). The human acid α-glucosidase fraction iseluted from the Q FF column with 2-3 cv buffer A, containing 100 mMNaCl.

The Q FF Sepharose human acid α-glucosidase containing fraction can befurther purified using Phenyl Sepharose High Performance chromatography.For example, 1 vol. of 1 M ammonium sulphate is added to the Q FFSepharose human acid α-glucosidase eluate while stirring continuously.Phenyl HP (Pharmacia) column chromatography (Pharmacia XK-50 column, 15cm bed height; 150 cm/hr flow rate) is then done at room temperature byequilibrating the column in 0.5 M ammonium sulphate, 50 mM sodiumphosphate buffer pH 6.0 (buffer C), loading the 0.5 Mammoniumsulphate-incubated human acid α-glucosidase eluate (from Q FFSepharose), washing the column with 24 cv of buffer C, and eluting thehuman acid α-glucosidase was eluted from the Phenyl HP column with 3-5cv buffer D (50 mM sodium phosphate buffer at pH 6.0). Alternativemethods and additional methods for further purifying human acidα-glucosidase will be apparent to those of skill. For example, seeUnited Kingdom patent application 998 07464.4 (incorporated by referencein its entirety for all purposes).

G. Uses of Recombinant Lysosomal Proteins

The recombinant lysosomal proteins produced according to the inventionfind use in enzyme replacement therapeutic procedures. A patient havinga genetic or other deficiency resulting in an insufficiency offunctional lysosomal enzyme can be treated by administering exogenousenzyme to the patient. Patients in need of such treatment can beidentified from symptoms (e.g., Hurler's syndrome symptoms includeDwarfism, corneal clouding, hepatosplenomegaly, valvular lesions,coronary artery lesions, skeletal deformities, joint stiffness andprogressive mental retardation). Alternatively, or additionally,patients can be diagnosed from biochemical analysis of a tissue sampleto reveal excessive accumulation of a characteristic metaboliteprocessed by a particular lysosomal enzyme or by enzyme assay using anartificial or natural substrate to reveal deficiency of a particularlysosomal enzyme activity. For most diseases, diagnosis can be made bymeasuring the particular enzyme deficiency or by DNA analysis beforeoccurrence of symptoms or excessive accumulation of metabolites (Scriveret al., supra, chapters on lysosomal storage disorders). All of thelysosomal storage diseases are hereditary. Thus, in offspring fromfamilies known to have members suffering from lysosomal diseases, it issometimes advisable to commence prophylactic treatment even before adefinitive diagnosis can be made.

Pharmaceutical Compositions

In some methods, lysosomal enzymes are administered in purified formtogether with a pharmaceutical carrier as a pharmaceutical composition.The preferred form depends on the intended mode of administration andtherapeutic application. The pharmaceutical carrier can be anycompatible, nontoxic substance suitable to deliver the polypeptides tothe patient. Sterile water, alcohol, fats, waxes, and inert solids maybe used as the carrier. Pharmaceutically-acceptable adjuvants, bufferingagents, dispersing agents, and the like, may also be incorporated intothe pharmaceutical compositions.

The concentration of the enzyme in the pharmaceutical composition canvary widely, i.e., from less than about 0.1% by weight, usually being atleast about 1% by weight to as much as 20% by weight or more.

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. Activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonateand the like. Examples of additional inactive ingredients that may beadded to provide desirable color, taste, stability, buffering capacity,dispersion or other known desirable features are red iron oxide, silicagel, sodium lauryl sulfate, titanium dioxide, edible white ink and thelike. Similar diluents can be used to make compressed tablets. Bothtablets and capsules can be manufactured as sustained release productsto provide for continuous release of medication over a period of hours.Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, orenteric-coated for selective disintegration in the gastrointestinaltract. Liquid dosage forms for oral administration can contain coloringand flavoring to increase patient acceptance.

A typical composition for intravenous infusion could be made up tocontain 100 to 500 ml of sterile 0.9% NaCl or 5% glucose optionallysupplemented with a 20% albumin solution and 100 to 500 mg of an enzyme.A typical pharmaceutical compositions for intramuscular injection wouldbe made up to contain, for example, 1 ml of sterile buffered water and 1to 10 mg of the purified alpha glucosidase of the present invention.Methods for preparing parenterally administrable compositions are wellknown in the art and described in more detail in various sources,including, for example, Remington's Pharmaceutical Science (15th ed.,Mack Publishing, Easton, Pa., 1980) (incorporated by reference in itsentirety for all purposes).

AGLU can be formulated in 10 mM sodium phosphate buffer pH 7.0. Smallamounts of ammonium sulphate are optionally present (<10 mM). The enzymeis typically kept frozen at about −70.degree. C., and thawed before use.Alternatively, the enzyme may be stored cold (e.g., at about 4.degree.C. to 8.degree. C.) in solution. In some embodiments, AGLU solutionscomprise a buffer (e.g., sodium phosphate, potassium phosphate or otherphysiologically acceptable buffers), a simple carbohydrate (e.g.,sucrose, glucose, maltose, mannitol or the like), proteins (e.g., humanserum albumin), and/or surfactants (e.g., polysorbate 80 (Tween-80),cremophore-EL, cremophore-R, labrofil, and the like).

AGLU can also be stored in lyophilized form. For lyophilization, AGLUcan be formulated in a solution containing mannitol, and sucrose in aphosphate buffer. The concentration of sucrose should be sufficient toprevent aggregation of AGLU on reconstitution. The concentration ofmannitol should be sufficient to significantly reduce the time otherwiseneeded for lyophilization. The concentrations of mannitol and sucroseshould, however, be insufficient to cause unacceptable hypertonicity onreconstitution. Concentrations of mannitol and sucrose of 1-3 mg/ml and0.1-1.0 mg/ml respectively are suitable. Preferred concentrations are 2mg/ml mannitol and 0.5 mg/ml sucrose. AGLU is preferably at 5 mg/mlbefore lyophilization and after reconstitution. Saline preferably at0.9% is a preferred solution for reconstitution.

For AGLU purified from rabbit milk, a small amount of impurities (e.g.,up to about 5%) can be tolerated. Possible impurities may be present inthe form of rabbit whey proteins. Other possible impurities arestructural analogues (e.g., oligomers and aggregates) and truncations ofAGLU. Current batches indicate that the AGLU produced in transgenicrabbits is >95% pure. The largest impurities are rabbit whey proteins,although on gel electrophoresis, AGLU bands of differing molecularweights are also seen.

Infusion solutions should be prepared aseptically in a laminar air flowhood. The appropriate amount of AGLU should be removed from the freezerand thawed at room temperature. Infusion solutions can be prepared inglass infusion bottles by mixing the appropriate amount of AGLU finishedproduct solution with an adequate amount of a solution containing humanserum albumin (HSA) and glucose. The final concentrations can be 1% HSAand 4% glucose for 25-200 mg doses and 1% HSA and 4% glucose for 400-800mg doses. HSA and AGLU can be filtered with a 0.2 . μm syringe filterbefore transfer into the infusion bottle containing 5% glucose.Alternatively, AGLU can be reconstituted in saline solution, preferably0.9% for infusion. Solutions of AGLU for infusion have been shown to bestable for up to 7 hours at room temperature. Therefore the AGLUsolution is preferably infused within seven hours of preparation.

Therapeutic Methods

The present invention provides effective methods of treating Pompe'sdisease. These methods are premised in part on the availability of largeamounts of human acid alpha glucosidase in a form that is catalyticallyactive and in a form that can be taken up by tissues, particularly,liver, heart and muscle (e.g., smooth muscle, striated muscle, andcardiac muscle), of a patient being treated. Such human acidalpha-glucosidase is provided from e.g., the transgenic animalsdescribed in the Examples. The alpha-glucosidase is preferablypredominantly (i.e., >50%) in the precursor form of about 100-110 kD.(The apparent molecular weight or relative mobility of the 100-110 kDprecursor may vary somewhat depending on the method of analysis used,but is typically within the range 95 kD and 120 kD.) Given thesuccessful results with human acid alpha-glucosidase in the transgenicanimals discussed in the Examples, it is possible that other sources ofhuman alpha-glucosidase, such as resulting from cellular expressionsystems, can also be used. For example, an alternative way to producehuman acid α-glucosidase is to transfect the acid α-glucosidase geneinto a stable eukaryotic cell line (e.g., CHO) as a cDNA or genomicconstruct operably linked to a suitable promoter. However, it is morelaborious to produce the large amounts of human acid alpha glucosidaseneeded for clinical therapy by such an approach.

The pharmaceutical compositions of the present invention are usuallyadministered intravenously. Intradermal, intramuscular or oraladministration is also possible in some circumstances. The compositionscan be administered for prophylactic treatment of individuals sufferingfrom, or at risk of, a lysosomal enzyme deficiency disease. Fortherapeutic applications, the pharmaceutical compositions areadministered to a patient suffering from established disease in anamount sufficient to reduce the concentration of accumulated metaboliteand/or prevent or arrest further accumulation of metabolite. Forindividuals at risk of lysosomal enzyme deficiency disease, thepharmaceutical compositions are administered prophylactically in anamount sufficient to either prevent or inhibit accumulation ofmetabolite. An amount adequate to accomplish this is defined as a“therapeutically-” or “prophylactically-effective dose.” Such effectivedosages will depend on the severity of the condition and on the generalstate of the patient's health.

In the present methods, human acid alpha glucosidase is usuallyadministered at a dosage of 10 mg/kg patient body weight or more perweek to a patient. Often dosages are greater than 10 mg/kg per week.Dosages regimes can range from 10 mg/kg per week to at least 1000 mg/kgper week. Typically dosage regimes are 10 mg/kg per week, 15 mg/kg perweek, 20 mg/kg per week, 25 mg/kg per week, 30 mg/kg per week, 35 mg/kgper week, 40 mg/kg week, 45 mg/kg per week, 60 mg/kg week, 80 mg/kg perweek and 120 mg/kg per week. In preferred regimes 10 mg/kg, 15 mg/kg, 20mg/kg, 30 mg/kg or 40 mg/kg is administered once, twice or three timesweekly. Treatment is typically continued for at least 4 weeks, sometimes24 weeks, and sometimes for the life of the patient. Treatment ispreferably administered i.v. Optionally, levels of humanalpha-glucosidase are monitored following treatment (e.g., in the plasmaor muscle) and a further dosage is administered when detected levelsfall substantially below (e.g., less than 20%) of values in normalpersons.

In some methods, human acid alpha glucosidase is administered at aninitially “high” dose (i.e., a “loading dose”), followed byadministration of a lower doses (i.e., a “maintenance dose”). An exampleof a loading dose is at least about 40 mg/kg patient body weight 1 to 3times per week (e.g., for 1, 2, or 3 weeks). An example of a maintenancedose is at least about 5 to at least about 10 mg/kg patient body weightper week, or more, such as 20 mg/kg per week, 30 mg/kg per week, 40mg/kg week.

In some methods, a dosage is administered at increasing rate during thedosage period. Such can be achieved by increasing the rate of flowintravenous infusion or by using a gradient of increasing concentrationof alpha-glucosidase administered at constant rate. Administration inthis manner reduces the risk of immunogenic reaction. In some dosages,the rate of administration measured in units of alpha glucosidase perunit time increases by at least a factor of ten. Typically, theintravenous infusion occurs over a period of several hours (e.g., 1-10hours and preferably 2-8 hours, more preferably 3-6 hours), and the rateof infusion is increased at intervals during the period ofadministration.

Suitable dosages (all in mg/kg/hr) for infusion at increasing rates areshown in table 1 below. The first column of the table indicates periodsof time in the dosing schedule. For example, the reference to 0-1 hrrefers to the first hour of the dosing. The fifth column of the tableshows the range of doses than can be used at each time period. Thefourth column shows a narrower included range of preferred dosages. Thethird column indicates upper and lower values of dosages administered inan exemplary clinical trial. The second column shows particularlypreferred dosages, these representing the mean of the range shown in thethird column of table 1.

TABLE 1 Lower & Upper Preferred Time Mean Doses (I) Values Range Range0-1 hr: 0.3 mg/kg/hr   0.25-0.4  0.1-1   0.03-3  1-2 hr: 1 mg/kg/hr0.9-1.4 1-4  0.3-12 2.2.5 hr: 4 mg/kg/hr 3.6-5.7 3-10   1-30 2.5-5.6 hr:12 mg/kg/hr   7.2-11.3 6-20 2.60

The methods are effective on patients with both early onset (infantile)and late onset (juvenile and adult) Pompe's disease. In patients withthe infantile form of Pompe's disease symptoms become apparent withinthe first 4 months of life. Mostly, poor motor development and failureto thrive are noticed first. On clinical examination, there isgeneralized hypotonia with muscle wasting, increased respiration ratewith sternal retractions, moderate enlargement of the liver, andprotrusion of the tongue. Ultrasound examination of the heart shows aprogressive hypertrophic cardiomyopathy, eventually leading toinsufficient cardiac output. The ECG is characterized by marked leftaxis deviation, a short PR interval, large QRS complexes, inverted Twaves and ST depression. The disease shows a rapidly progressive courseleading to cardiorespiratory failure within the first year of life. Onhistological examination at autopsy lysosomal glycogen storage isobserved in various tissues, and is most pronounced in heart andskeletal muscle. Treatment with human acid alpha glucosidase in thepresent methods results in a prolongation of life of such patients(e.g., greater than 1, 2, 5 years up to a normal lifespan). Treatmentcan also result in elimination or reduction of clinical and biochemicalcharacteristics of Pompe's disease as discussed above. Treatment isadministered soon after birth, or antenatally if the parents are knownto bear variant alpha glucosidase alleles placing their progeny at risk.

Patients with the late onset adult form of Pompe's disease may notexperience symptoms within the first two decades of life. In thisclinical subtype, predominantly skeletal muscles are involved withpredilection of those of the limb girdle, the trunk and the diaphragm.Difficulty in climbing stairs is often the initial complaint. Therespiratory impairment varies considerably. It can dominate the clinicalpicture, or it is not experienced by the patient until late in life.Most such patients die because of respiratory insufficiency. In patientswith the juvenile subtype, symptoms usually become apparent in the firstdecade of life. As in adult Pompe's disease, skeletal muscle weakness isthe major problem; cardiomegaly, hepatomegaly, and macroglossia can beseen, but are rare. In many cases, nightly ventilatory support isultimately needed. Pulmonary infections in combination with wasting ofthe respiratory muscles are life threatening and mostly become fatalbefore the third decade. Treatment with the present methods prolongs thelife of patients with late onset juvenile or adult Pompe's disease up toa normal life span. Treatment also eliminates or significantly reducesclinical and biochemical symptoms of disease.

Other Uses

Lysosomal proteins produced in the milk of transgenic animals have anumber of other uses. For example, α-glucosidase, in common with otherα-amylases, is an important tool in production of starch, beer andpharmaceuticals. See. Vihinen and Mantsala, Crit. Rev. Biochem. Mol.Biol. 24, 329-401 (1989) (incorporated by reference in its entirety forall purpose). Lysosomal proteins are also useful for producinglaboratory chemicals or food products. For example, acid α-glucosidasedegrades 1,4 and 1,6 α-glucidic bonds and can be used for thedegradation of various carbohydrates containing these bonds, such asmaltose, isomaltose, starch and glycogen, to yield glucose. Acidα-glucosidase is also useful for administration to patients with anintestinal maltase or isomaltase deficiency. Symptoms otherwiseresulting from the presence of undigested maltose are avoided. In suchapplications, the enzyme can be administered without prior fractionationfrom milk, as a food product derived from such milk (e.g., ice cream orcheese) or as a pharmaceutical composition. Purified recombinantlysosomal enzymes are also useful for inclusion as controls indiagnostic kits for assay of unknown quantities of such enzymes intissue samples.

EXAMPLES Example 1 Construction of Transgenes

(a) cDNA Construct

Construction of an expression vector containing cDNA encoding human acidα-glucosidase started with the plasmid p16,8hlf3 (see DeBoer et al.(1991) and (1993), supra) This plasmid includes bovine αS1-caseinregulatory sequences. The lactoferrin cDNA insert of the parent plasmidwas exchanged for the human acid α-glucosidase cDNA (Hoefsloot et al.EMBO J. 7, 1697-1704 (1988)) at the ClaI site and SalI site of theexpression cassette as shown in FIG. 1. To obtain the compatiblerestriction sites at the ends of the α-glucosidase cDNA fragment,plasmid pSHAG2 (id.) containing the complete cDNA encoding humanα-glucosidase was digested with EcoRI and SphI and the 3.3 kbcDNA-fragment was subcloned in pKUN7ΔC, a pKUN1 derivative (Konings etal., Gene 46, 269-276 (1986)), with a destroyed ClaI site within thevector nucleotide sequences and with a newly designed polylinker:HindIII ClaI EcoRI SphI XhoI EcoRI SflI SfiI/SmaI NotIEcoRI*(*=destroyed site). From the resulting plasmid pagluCESX, the3.3-kb cDNA-fragment could be excised by ClaI and XhoI. This fragmentwas inserted into the expression cassette shown in FIG. 1 at the ClaIsite and XhoI-compatible SalI site. As a result, the expression plasmidp16,8αglu consists of the cDNA sequence encoding human acidα-glucosidase flanked by bovine αS1 casein sequences as shown in FIG. 1.The 27.3-kb fragment containing the complete expression cassette can beexcised by cleavage with NotI (see FIG. 1).

(b) Genomic Constructs

Construct c8αgluex1 contains the human acid α-glucosidase gene(Hoefsloot et al., Biochem. J. 272, 493-497 (1990)); starting in exon 1just downstream of its transcription initiation site (see FIG. 2, panelA). Therefore, the construct encodes almost a complete 5′ UTR of thehuman acid α-glucosidase gene. This fragment was fused to the promotersequences of the bovine αS1-casein gene. The αS1-casein initiation siteis present 22 by upstream of the αS1-casein/acid α-glucosidase junction.The construct has the human acid α-glucosidase polyadenylation signaland 3′ flanking sequences. Construct c8αgluex2 contains the bovineαS1-casein promoter immediately fused to the translation initiation sitein exon 2 of the human acid α-glucosidase gene (see FIG. 2, panel B).Thus, the αS1-casein transcription initiation site and the α-glucosidasetranslation initiation site are 22-bp apart in this construct. Thereforeno α-glucosidase 5′ UTR is preserved. This construct also contains thehuman acid α-glucosidase polyadenylation signal and 3′ flankingsequences as shown in FIG. 2, panel B.

Construct c8,8αgluex2-20 differs from construct c8αgluex2 only in the 3′region. A SphI site in exon 20 was used to fuse the bovine αS1-casein 3′sequence to the human acid α-glucosidase construct. The polyadenylationsignal is located in this 3′ αS1-casein sequence (FIG. 2, panel C).

Construct c8,8αgluex2-20 differs from construct c8αgluex2 only in the 3′region. A SphI site in exon 20 was used to fuse the bovine αS1-casein 3′sequence to the human acid α-glucosidase construct. The polyadenylationsignal is located in this 3′ αS1 casein sequence (FIG. 2, panel C).

Methods for Construction of Genomic Constructs

Three contiguous BglII fragments containing the human acid α-glucosidasegene were isolated by Hoefsloot et al., supra. These fragments wereligated in the BglII-site of pKUN8ΔC, a pKUN7ΔC derivative with acustomized polylinker: HindIII ClaI StuI SstI BglII PvnI NcoI EcoRI SphIXhoI EcoRI* SmaI/SfiI NotI EcoRI* (*=destroyed site). This ligationresulted in two orientations of the fragments generating plasmidsp7.3αgluBES, p7.3αgluBSE, p8.5αgluBSE, p8.5αgluBES, p10αgluBSE andp10αgluBES.

Because unique NotI-sites at the ends of the expression cassette areused subsequently for the isolation of the fragments used formicroinjection, the intragenic NotI site in intron 17 of human acidα-glucosidase had to be destroyed. Therefore, p10αgluBES was digestedwith ClaI and XhoI; the fragment containing the 3′ α-glucosidase end wasisolated. This fragment was inserted in the ClaI and XhoI sites ofpKUN10ΔC, resulting in p10αgluNV. Previously pKUN10ΔC (i.e., aderivative of pKUN8ΔC) was obtained by digesting pKUN8ΔC with NotI,filling in the sticky ends with Klenow and subsequently, annealing theplasmid by blunt-ended ligation. Finally, p10αgluΔNV was digested withNotI. These sticky ends were also filled with Klenow and the fragmentwas ligated, resulting in plasmid p10αgluΔNotI.

Construction of c8αgluex1

Since the SstI site in first exon of the α-glucosidase gene was chosenfor the fusion to the bovine αS1-casein sequence, p8.5αgluBSE wasdigested with BglII followed by a partial digestion with SstI. Thefragment containing exon 1-3 was isolated and ligated into the BglII andSstI sites of pKUN8ΔC. The resulting plasmid was named: p5′αgluex1. (seeFIG. 3, panel A).

The next step (FIG. 3, panel B) was the ligation of the 3′ part top5′αgluex1. First, p10αgluΔN was digested with BglII and BamHI. Thisfragment containing exon 16-20 was isolated. Second, p5′αgluex1 wasdigested with BglII and to prevent self-ligation, and treated withphosphorylase (BAP) to dephosphorylate the sticky BglII ends. SinceBamHI sticky ends are compatible with the BglII sticky ends, these endsligate to each other. The resulting plasmid, i.e., p5′3′αgluex1, wasselected. This plasmid has a unique BglII site available for the finalconstruction step (see FIG. 3, panels B and C).

The middle part of the α-glucosidase gene was inserted into the latterconstruct. For this step, p7.3αgluBSE was digested with BglII, the8.5-kb fragment was isolated and ligated to the BglII digested anddephosphorylated p5′3′αgluex1 plasmid. The resulting plasmid is pαgluex1(FIG. 3, panel C).

The bovine αS1-casein promoter sequences were incorporated in the nextstep via a ligation involving three fragments. The pWE15 cosmid vectorwas digested with NotI and dephosphorylated. The bovine αS1-caseinpromoter was isolated as an 8 Rb NotI-ClaI fragment (see de Boer et al.,1991, supra). The human acid α-glucosidase fragment was isolated frompαgluex1 using the same enzymes. These three fragments were ligated andpackaged using the Stratagene GigapackII kit in 1046 E. coli host cells.The resulting cosmid c8αgluex1 was propagated in E. coli strain DH5α.The vector was linearized with NotI before microinjection.

Construction of c8αgluex2 and c8.8αgluex2-20

The construction of the other two expression plasmids (FIG. 2, panels Band C) followed a similar strategy to that of c8αgluex1. The plasmidp5′αgluStuI was derived from p8,5αgluBSE by digestion of the plasmidwith StuI, followed by self-ligation of the isolated fragment containingexon 2-3 plus the vector sequences. Plasmid p5′αgluStuI was digestedwith PglII followed by a partial digestion of the linear fragment withNcoI resulting in several fragments. The 2.4 kb fragment, containingexon 2 and 3, was isolated and ligated into the NcoI and BglII sites ofvector pKUN12ΔC, resulting in p5′αgluex2. Note that pKUN12ΔC is aderivative of pKUN8ΔC containing the polylinker: ClaI NcoI BglII HindiEcoRI SphI XhoI SmaI/SfiI NotI.

The plasmid p10αgluΔNotI was digested with BglII and HindIII. Thefragment containing exons 16-20 was isolated and ligated in p5′αgluex2digested with BglII and HindIII. The resulting plasmid was p5′3′αgluex2.The middle fragment in p5′3′αgluex2 was inserted as for pαgluex1. Forthis, p7.3αglu was digested with BglII. The fragment was isolated andligated to the BglII-digested and dephosphorylated p5′3′αgluex2. Theresulting plasmid, pαgluex2, was used in construction of c8αgluex-20 andc8,8αgluex2-20 (FIG. 2, panels B and C).

For the construction of third expression plasmid c8,8α gluex2-20 (FIG.2, panel C) the 3′ flanking region of α-glucosidase was deleted. Toachieve this, pαgluex2 was digested with SphI. The fragment containingexon 2-20 was isolated and self-ligated resulting in pαgluex2-20.Subsequently, the fragment containing the 3′ flanking region of bovineαS1-casein gene was isolated from p16,8αglu by digestion with SphI andNotI. This fragment was inserted into pαgluex2-20 using the SpII siteand the NotI site in the polylinker sequence resulting inpαgluex2-20-3αS1.

The final step in generating c8,8αgluex2-20 was the ligation of threefragments as in the final step in the construction leading to c8αgluex1.Since the ClaI site in pαgluex2-20-3′αS1 and pαgluex2 appeared to beuncleavable due to methylation, the plasmids had to be propagated in theE. coli DAM(−) strain ECO343. The pαgluex2-20-3′αS1 isolated from thatstrain was digested with ClaI and NotI. The fragment containing exons220 plus the 3′αS1-casein flanking region was purified from the vectorsequences. This fragment, an 8 kb NotI-ClaI fragment containing thebovine αS1 promoter (see DeBoer (1991) and (1993), supra) andNotI-digested, dephosphorylated pWE15 were ligated and packaged. Theresulting cosmid is c8,8αgluex2-20.

Cosmid c8αgluex2 (FIG. 2, panel B) was constructed via a couple ofdifferent steps. First, cosmid c8,8αgluex2-20 was digested with SalI andNotI. The 10.5-kb fragment containing the αS1-promoter and the exons 2-6part of the acid α-glucosidase gene was isolated. Second, plasmidpαgluex2 was digested with SalI and NotI to obtain the fragmentcontaining the 3′ part of the acid α-glucosidase gene. Finally, thecosmid vector pWE15 was digested with NotI and dephosphorylated. Thesethree fragments were ligated and packaged. The resulting cosmid isc8αgluex2.

Example 2 Transgenesis

The cDNA and genomic constructs were linearized with NotI and injectedin the pronucleus of fertilized mouse oocytes which were then implantedin the uterus of pseudopregnant mouse foster mothers. The offspring wereanalyzed for the insertion of the human α-glucosidase cDNA or genomicDNA gene construct by Southern blotting of DNA isolated from clippedtails. Transgenic mice were selected and bred.

The genomic constructs linearized with NotI were also injected into thepronucleus of fertilized rabbit oocytes, which were implanted in theuterus of pseudopregnant rabbit foster mothers. The offspring wereanalyzed for the insertion of the alpha-glucosidase DNA by Southernblotting. Transgenic rabbits were selected and bred.

Example 3 Analysis of Acid α-Glucosidase in the Milk of Transgenic Mice

Milk from transgenic mice and nontransgenic controls was analyzed byWestern Blotting. The probe was mouse antibody specific for human acidα-glucosidase (i.e., does not bind to the mouse enzyme). Transgenes 1672and 1673 showed expression of human acid α-glucosidase in milk (FIGS. 4Aand 4B). Major and minor bands at 100-110 kD and 76 kD were observed asexpected for α-glucosidase. In milk from non-transgenic mice, no bandswere observed.

The activity of human acid α-glucosidase was measured with theartificial substrate 4-methylumbelliferyl-α-D-glucopyranoside in themilk of transgenic mouse lines (See Galiaard, Genetic Metabolic Disease,Early Diagnosis and Prenatal Analysis, Elsevier/North Holland,Amsterdam, pp. 809-827 (1980)). Mice containing the cDNA construct(FIG. 1) varied from 0.2 to 2 . μmol/ml per hr. The mouse linescontaining the genomic construct (FIG. 2, panel A) expressed at levelsfrom 10 to 610 . μmol/ml per hr. These figures are equivalent to aproduction of 1.3 to 11.3 mg/l (cDNA construct) and 0.05 to 3.3 g/l(genomic construct) based on an estimated specific activity of therecombinant enzyme of 180 . μmol/mg (Van der Ploeg et al., J. Neurol.235:392-396 (1988)).

The recombinant acid α-glucosidase was isolated from the milk oftransgenic mice, by sequential chromatography of milk on ConA-Sepharose™and Sephadex™ G200. 7 ml milk was diluted to 10 ml with 3 ml Con Abuffer consisting of 10 mM sodium phosphate, pH 6.6 and 100 mM NaCl. A1:1 suspension of Con A sepharose in Con A buffer was then added and themilk was left overnight at 4.degree. C. with gentle shaking. The Con Asepharose beads were then collected by centrifugation and washed 5 timeswith Con A buffer, 3 times with Con A buffer containing 1 M NaCl insteadof 100 mM, once with Con A buffer containing 0.5 M NaCl instead of 100mM and then eluted batchwise with Con A buffer containing 0.5 M NaCl and0.1 M methyl-α-D-mannopyranoside. The acid α-glucosidase activity in theeluted samples was measured using the artificial4-methyl-umbelliferyl-α-D-glycopyranoside substrate (see above).Fractions containing acid α-glucosidase activity were pooled,concentrated and dialyzed against Sephadex buffer consisting of 20 mM Naacetate, pH 4.5 and 25 mM NaCl, and applied to a Sephadex™ 200 column.This column was run with the same buffer, and fractions were assayed foracid α-glucosidase activity and protein content. Fractions rich in acidα-glucosidase activity and practically free of other proteins werepooled and concentrated. The method as described is essentially the sameas the one published by Reuser et al., Exp. Cell Res. 155:178-179(1984). Several modifications of the method are possible regarding theexact composition and pH of the buffer systems and the choice ofpurification steps in number and in column material.

Acid α-glucosidase purified from the milk was then tested forphosphorylation by administrating the enzyme to cultured fibroblastsfrom patients with GSD II (deficient in endogenous acid α-glucosidase).In this test mannose 6-phosphate containing proteins are bound bymannose 6-phosphate receptors on the cell surface of fibroblasts and aresubsequently internalized. The binding is inhibited by free mannose6-phosphate (Reuser et al., Exp. Cell Res. 155:178-189 (1984)). In atypical test for the phosphorylation of acid α-glucosidase isolated frommilk of transgenic mice, the acid α-glucosidase was added to 104-106fibroblasts in 500 . μl culture medium (Ham F10, supplied with 10% fetalcalf serum and 3 mM Pipes) in an amount sufficient to metabolize 1 mmole4-methyl-umbelliferyl-α-D-glucopyranoside per hour for a time period of20 hours. The experiment was performed with or without 5 mM mannose6-phosphate as a competitor, essentially as described by Reuser et al.,supra (1984). Under these conditions acid α-glucosidase of the patientfibroblasts was restored to the level measured in fibroblasts fromhealthy individuals. The restoration of the endogenous acidα-glucosidase activity by acid α-glucosidase isolated from mouse milkwas as efficient as restoration by acid α-glucosidase purified frombovine testis, human urine and medium of transfected CHO cells.Restoration by α-glucosidase form milk was inhibited by 5 mM mannose6-phosphate as observed for α-glucosidase from other sources. (Reuser etal., supra; Van der Ploeg et al., (1999), supra; Van der Ploeg et al.,Ped. Res. 24:90-94 (198B).

As a further demonstration of the authenticity of α-glucosidase producedin the milk, the N-terminal amino acid sequence of the recombinantα-glucosidase produced in the milk of mice was shown to be the same asthat of α-glucosidase precursor from human urine as published byHoefsloot et al., EMBO J. 7:1697-1704 (1988) which starts with AHPGRP(SEQ ID NO:1).

Example 4 Animal Trial of Alpha-Glucosidase

Recently, a knock-out mouse model for Pompe's disease has becomeavailable (25) This model was generated by targeted disruption of themurine alpha-glucosidase gene. Glycogen-containing lysosomes aredetected soon after birth in liver, heart and skeletal muscle. Overtclinical symptoms only become apparent at relatively late age (>9months), but the heart is typically enlarged and the electrocardiogramis abnormal.

Experiments have been carried out using the knock-out (KO) mouse modelin order to study the in vivo effect of AGLU purified from transgenicrabbit milk. The recombinant enzyme in these experiments was purifiedfrom milk of the transgenic rabbits essentially as described above forpurification from transgenic mice.

1. Short Term Studies in KO Mouse Model

Single or multiple injections with a 6 day interval were administered toKO mice via the tail vein. Two days after the last enzyme administrationthe animals were killed, and the organs were perfused with phosphatebuffered saline (PBS). Tissue homogenates were made for GLU enzymeactivity assays and tissue glycogen content, and ultrathin sections ofvarious organs were made to visualize accumulation (via electronmicroscopy) lysosomal glycogen content. Also the localization ofinternalized AGLU was determined using rabbit polyclonal antibodiesagainst human placenta mature α-glucosidase.

The results showed that single doses of 0.7 and 1.7 mg AGLU (experimentsC and A respectively) was taken up efficiently in vivo in various organsof groups of two knock-out mice when injected intravenously. ExperimentA also showed that there were no differences in the uptake anddistribution of AGLU purified from two independent rabbit milk sources.

Increases in AGLU activity were seen in the organs such as the liver,spleen, heart, and skeletal muscle, but not in the brain. Two days aftera single injection of 1.7 mg AGLU to two KO animals, levels close to, ormuch higher than, the endogenous alpha-glucosidase activity levelsobserved in organs of two PBS-injected normal control mice or twoheterozygous KO mice were obtained (experiment A). Of the organs tested,the liver and spleen are, quantitatively, the main organs involved inuptake, but also the heart and pectoral and femoral muscles take upsignificant amounts of enzyme. The absence of a significant increase inbrain tissue suggests that AGLU does not pass the blood-brain barrier.The results are summarized in Table 2.

TABLE 2 Tissue Uptake of AGLU and Glycogen Content Following Short TermTreatment in KO Mouse Model Pectoral Femoral Liver Spleen Heart MuscleMuscle Tongue Brain Group Act Glc Act Glc Act Glc Act Glc Act Glc ActGlc Act Glc Experiment A animals treated with single dose of 1.7 mg AGLU(from 2 sources) treated KO 674 — — — 263 — — — 24 — — — 0.8 — micesource 1 410 17 3.1 0.4 treated KO 454 — — — 76 — — — 12 — — — 0.8 —mice source 2 604 48 10 0.4 untreated KO 3.1 — — — 0.2 — — — 0.2 — — —0.2 — mouse untreated 58 — — — 23 — — — 11 — — — 57 — normal mouse 37 178.2 57 Experiment B animals treated with 4 doses of AGLU (1.0, 2.0, 1.0and 1.4 mg.) 6 days apart treated KO 1132 70 — — 24 1259 125 87 — — 89 —0.4 163 mice (13 944 13 10 1082 46 116 35 0.2 163 weeks old) treated KO3375 23 — — 60 1971 49 90 — — 207 — 0.7 374 mice (34 weeks old)untreated KO 2.0 406 — — 0.2 3233 1.0 86 — — 1.0 — 0.2 487 mice (13 and2.0 147 0.3 1748 1.0 87 1.0 0.2 168 34 weeks old) untreated 35 6 — — 8.20 6.0 1.0 — — 14 — 18 0 normal mice (34 weeks old) treated KO 582 — 462— 46 — — — 5.1 — — — 0.4 — mice 558 313 50 3.6 0.4 untreated KO 1.1 —0.8 — 0.3 — — — 0.2 — — — 0.2 — mice 1.6 0.7 0.3 0.3 0.2 Figures in thetable refer to individual animals Act: AGLU activity (nmoles 4MU per mgprotein per hour) GLC: Glycogen content (μg/mg protein) n.d. notdetected — data unavailable

When two KO mice were injected 4 times every 6 days (experiment B), amarked decrease of total cellular glycogen was observed in both heartand liver. No effects were observed in skeletal muscle tissues withregard to total glycogen. However, in general the uptake of AGLU inthese tissues was lower than in the other tissues tested.

Transmission electron microscopy of the 4 times injected KO miceindicated a marked decrease in lysosomal glycogen in both liver cellsand heart muscle cells. The effects observed in heart tissue arelocalized since in some areas of the heart no decrease in lysosomalglycogen was observed after these short term administrations.

Western blot analysis using rabbit polyclonal antibodies against humanplacenta mature alpha-glucosidase indicated complete processing of theinjected AGLU towards the mature enzyme in all organs tested stronglysuggesting uptake in target tissues, and lysosomal localization andprocessing. No toxic effects were observed in any of the threeexperiments.

Immunohistochemical staining of AGLU was evident in lysosomes ofhepatocytes using a polyclonal rabbit antibody against humanalpha-glucosidase. The presence of AGLU in heart and skeletal tissues ismore difficult to visualize with this technique, apparently due to thelower uptake.

2. Long-Term Experiments with the KO Mouse Model

In longer term experiments, enzyme was injected in the tail vein ofgroups of two or three KO mice, once a week for periods of up to 25weeks. The initial dose was 2 mg (68 mg/kg) followed by 0.5 mg (17mg/kg)/mouse for 12 weeks. In two groups of mice, this was followed byeither 4 or 11 additional weeks of treatment of 2 mg/mouse. Injectionsstarted when the mice were 6-7 months of age. At this age, clearhistopathology has developed in the KO model. Two days after the lastenzyme administration the animals were killed, and the organs wereperfused with phosphate buffered saline (PBS). Tissue homogenates weremade for AGLU enzyme activity assays and tissue glycogen content, andsections of various organs were made to visualize (via light microscopy)lysosomal glycogen accumulation.

The results showed that mice treated 13 weeks with 0.5 mg/mouse (GroupA, 3 animals/Group) had an increase of activity in the liver and spleenand decreased levels of glycogen in liver and perhaps in heart. Oneanimal showed increased activity in muscles, although there was nosignificant decrease of glycogen in muscle.

Mice that were treated 14 weeks with 0.5 mg/mouse followed by 4 weekswith 2 mg/mouse (Group B, 3 animals/Group) showed similar results tothose treated for 13 weeks only, except that an increased activity wasmeasured in the heart and skeletal muscles and decreases of glycogenlevels were also seen in the spleen.

Mice that were treated 14 weeks with 0.5 mg/mouse followed by 11 weekswith 2 mg/mouse (Group C 2 animals/Group) showed similar results to theother two groups except that treated mice showed definite decreases inglycogen levels in liver, spleen, heart and skeletal muscle. No activitycould be detected, even at the highest dose, in the brain.

Results of treated and untreated animals in each Group (Group means) aresummarized in Table 3.

TABLE 3 Tissue Uptake of AGLU and Glycogen Content Following Long TermTreatment in KO Mouse Model Pectoral Quadriceps Gastroenemius LiverSpleen Heart Muscle Muscle Muscle Brain Group Act Glc Act Glc Act GlcAct Glc Act Glc Act Glc Act Glc Group A animals treated with 0.5mg/mouse/week for 13 weeks treated 713 2 463 n.d. 3 86 9 81 6 40 14 66 —— untreated 2 24 1 n.d. 1 111 1 66 1 1 61 — — Group B animals treatedwith 0.5 mg/mouse/week for 14 weeks, followed by 2 mg/mouse/week for 4weeks treated 2705 1 1628 0 59 288 49 120 30 128 44 132 — — untreated 311 31 6 1 472 1 113 1 162 1 142 — — Group C animals treated with 0.5mg/mouse/week for 14 weeks, followed by 2 mg/mouse/week for 11 weekstreated 1762 1 1073 2 66 211 99 113 37 18 109 32 1 32 untreated 2 45 121 1 729 1 291 0 104 0 224 0 44 Figures in the table refer to the meanof 3animals (Groups A and B) or the mean of 2 animals (Group C) Act:AGLU activity (nmoles 4MU per mg protein per hour) GLC: Glycogen content(μg/mg protein) n.d. not detected — data unavailable

In addition, a very convincing improvement in the histopathologicalcondition was observed in Group C mice (treated for the first 14 weeksat 0.5 mg/mouse, followed by 11 weeks at 2 mg/mouse). Clear reversal ofpathology was demonstrated in various tissues, such as heart andpectoralis muscle.

It has been reported that Pompe's disease does not occur when theresidual lysosomal α-glucosidase activity is >20% of average controlvalue (14). The data obtained with the KO mouse model indicates thatthese levels are very well achievable using recombinant precursorenzyme.

Example 5 Human Clinical Trial

A single phase I study (AGLUI 101-01) has been conducted in 15 healthymale volunteers. Doses of AGLU ranged from 25 to 800 mg, administered byintravenous infusion to healthy male adult volunteers. Subjects with ahistory of allergies and hypersensitivities were excluded from thestudy. The subjects were randomized into dose groups of 5, and each doseGroup received AGLU (4 subjects) or placebo (1 subject) at each doselevel. All subjects received two doses of study drug, which wereadministered two weeks apart. The dosing regimen was as follows:

A

25 mg: Group 1, treatment period 1

B

50 mg: Group 1, treatment period 2

C

100 mg: Group 2, treatment period 1

D

200 mg: Group 3, treatment period 1

E

400 mg: Group 2, treatment period 2

F

800 mg: Group 3, treatment period 2

P

placebo (1 subject per Group and treatment period)

Subjects were administered AGLU or placebo as an infusion on Day 1 ofeach treatment period. The infusions were administered over a 30 minuteperiod and subjects were kept in a semi-recumbent position for at least2 hours after cessation of infusion.

Adverse events were recorded just before the start of the infusion, at30 minutes (end of infusion) and at 3, 12, 24, 36 and 48 hoursthereafter as well as on Days 5 and 8 (first period) and days 5, 8 and15 (second period). Vital signs, ECG and physical examinations were alsomonitored regularly throughout the treatment period.

Blood samples were taken for a standard range of clinical laboratorytests and pharmacokinetics analysis. The subject's urine was collectedand a standard range of laboratory analyses (including determination ofAGLU) were performed.

(a) Laboratory Safety and Adverse Events

There were no clinically significant changes in laboratory parameters,clinical signs and ECG measurements in any subjects at any dose Group.The results of adverse event monitoring in all subjects at all doses aresummarized in Table 4.

TABLE 4 Adverse Event Reports Dose (mg) Adverse Events 25 The reportedevents were muscle weakness, abnormal vision and fatigue. All eventswere mild and were deemed unrelated to the test article by theinvestigator 50 The reported events were headache, rhinitis, nose bleedand paresthesia. All events were mild and were deemed unrelated orremotely related to the test article by the investigator, except theparesthesia which was classed as moderate and was deemed possiblyrelated to the test article. 100 The reported events were rhinitis,headache, fatigue, hematoma and injection site reaction. All events wereclassed as mild. The cases of hematoma, injection site reaction andintermittent headache were deemed possibly or probably related to thetest article by the investigator. The other events were deemed to beunrelated. 200 The reported events were nausea, headache, dizziness,fatigue, rhinitis, photophobia, vision abnormalities and euphoria. Allevents were classed as mild or moderate in intensity. Seven events(including cases of dizziness, nausea and abnormal vision) were deemedto be possibly or probably related to the test article. 400 The reportedevents were fatigue and paresthesia. The report of fatigue wasconsidered unrelated to the test article, and the paresthesia was deemedpossibly related. 800 The reported events were nausea, drowsiness,dizziness, increased sweating, asthenia, shivering and intermittentheadache. All events were classed as mild or moderate in intensity.Eight events (including cases of drowsiness, nausea and asthenia) weredeemed to be possibly related to the test article.

A trial of the safety and efficacy of recombinant acid α-glucosidase asenzyme replacement therapy on infantile and juvenile patients withglycogen storage disease Type II is conducted. Four infantile patientsand three juvenile patients are recruited. Infantiles are administered astarting dose of 15-20 mg/kg titrated to 40 mg/kg and juveniles areadministered 10 mg/kg. Patients are treated for 24 weeks.

Patients are evaluated by the following parameters.

-   -   Standard adverse event reporting including suspected adverse        events    -   Laboratory parameters including hematology, clinical chemistry        and antibody detection.    -   α-glucosidase activity in muscle    -   Muscle histopathology    -   12-lead ECG    -   Clinical condition including neurological examination    -   Non-parametric PK parameters    -   Life saving interventions        Infantile patients are evaluated for the following additional        parameters.    -   Left posterior ventricular wall thickness and left ventricular        mass index    -   Neuromotor development    -   Survival    -   Glycogen content in muscle        Juvenile patients are evaluated for the following additional        parameters.    -   Pulmonary function    -   Muscle strength/timed tests and muscle function    -   PEDI/Rotterdam 9-item scale

The same patients are then subject to additional dosages of alphaglucosidase with infantiles receiving 15, 20, 30 or 40 mg/kg andjuveniles: 10 mg/kg for an additional period of 24 weeks and evaluatedby the parameters indicated above.

A further phase II clinical trial is performed on eight patients aged <6months of age within 2 months after diagnosis at a dosage of 40 mg/kg.Patients are treated for 24 weeks and evaluated by the followingcriteria:

Safety parameters

Laboratory safety data

Adverse event recording

Primary efficacy parameter: survival without life-saving interventions(i.e. mechanical ventilation >24 hr) 6 months past diagnosis incombination with normal or mildly delayed motor function (BSID II).

Secondary efficacy: Changes in neuromotor development, changes in leftposterior ventricular wall thickness and left ventricular mass index;Changes in skeletal muscle acid α-glucosidase activity and glycogencontent.

Efficacy can be show by a 50% survival at 6 months post-diagnosiswithout life saving interventions in the α-glucosidase group compared to10% survival in the historical control group in combination with a BSIDII classified as normal or mildly delayed.

A further clinical trial is performed on juvenile patients. The patientsare aged >1 year and <35 years of age with juvenile onset of GSD typeIIb The patients are administered 10 mg/kg or 20 mg/kg for a period oftwenty-four weeks treatment. Treatment is monitored by the followingparameters.

Safety parameters Laboratory safety data Adverse event recording Primaryefficacy Pulmonary function parameters (e.g. FVC, time on ventilator)Muscle strength Secondary efficacy Life-saving interventions parameters:Quality of life Skeletal muscle acid α-glucosidase activity Quantitativeobjective 20% relative improvement in primary efficacy parameters overbaselineAll quantitative measurements relating to efficacy are preferablystatistically significant relative to contemporaneous or historicalcontrols, preferably at p<0.05.

Example 6 Pharmaceutical Formulations

Alpha-glucosidase is formulated as follows: 5 mg/ml α-Glu, 15 mM sodiumphosphate, pH 6.5, 2% (w/w) mannitol, and 0.5% (w/w) sucrose. The aboveformulation is filled to a final volume of 10.5 ml into a 20 cc tubingvial and lyophilized. For testing, release and clinical use, each vialis reconstituted with 10.3 ml* of sterile saline (0.9%) for injection(USP or equivalent.) to yield 10.5 ml of a 5 mg/ml α-Glu solution thatmay be directly administered or subsequently diluted with sterile salineto a patient specific target dose concentration. The 10.5 ml fill (52.5mg alpha glucosidase total in vial) includes the USP recommendedoverage, that allows extraction and delivery (or transfer) of 10 mls (50mg). The mannitol serves as a suitable bulling agent shortening thelyophilization cycle (relative to sucrose alone). The sucrose serves asa cryo/lyoprotectant resulting in no significant increase in aggregationfollowing reconstitution. Reconstitution of the mannitol (only)formulations had repeatedly resulted in a slight increase inaggregation. Following lyophilization, the cake quality was acceptableand subsequent reconstitution times were significantly reduced Saline ispreferred to HSA/dextrose for infusion solution. When saline is used incombination with lyophilization in 2% mannitol/0.5% sucrose the solutionhas acceptable tonicity for intravenous administration. The lyophilizedvials containing the 2% mannitol/0.5% sucrose formulation werereconstituted with 0.9% sterile saline (for injection) to yield 5 mg/mlα-Glu.

Example 7 Infusion Schedule

The solution is administered via the indwelling intravenous cannula.Patients are monitored closely during the infusion period andappropriate clinical intervention are taken in the event of an adverseevent or suspected adverse event. A window of 48 hours is allowed foreach infusion. An infusion schedule in which the rate of infusionincreases with time reduces or eliminates adverse events.

Infusions for infantiles can be administered according to the followingschedule:

5 cc/hr for 60 minutes

10 cc/hr for 60 minutes

gtoreq.40 cc/hr for 30 minutes

gtoreq.80 cc/hr for the remainder of the infusion

Infusions for juveniles can be administered according to the followingschedule:

0.5 cc/kg/hr for 60 minutes

1 cc/kg/hr for 60 minutes

5 cc/kg/hr for 30 minutes

7.5 cc/kg hr for the remainder of the infusion

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. All publications and patent documents cited in thisapplication are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

1. A pharmaceutical composition comprising human acid alpha glucosidaseand a simple carbohydrate in a physiologically acceptable buffer insterile form.
 2. The pharmaceutical composition of claim 1, wherein thesimple carbohydrate is mannitol and the buffer is a sodium phosphatebuffer.
 3. The pharmaceutical composition of claim 2, wherein theconcentration of mannitol is 1-3% w/w of the solution.
 4. Thepharmaceutical composition of claim 2, wherein the concentration ofmannitol is 2% w/w of the solution.
 5. The pharmaceutical composition ofclaim 1, further comprising a surfactant.
 6. The pharmaceuticalcomposition of claim 5, wherein the surfactant is polysorbate
 80. 7. Apharmaceutical composition comprising alpha glucosidase and mannitol inan aqueous solution.
 8. The pharmaceutical composition of claim 7,wherein the human acid alpha-glucosidase is at 5 mg/ml and the mannitolis at 2 mg/ml
 9. A lyophilized composition produced by lyophilizing thepharmaceutical composition according claim
 1. 10. The lyophilizedcomposition of claim 9, further comprising a surfactant.
 11. Thelyophilized composition of claim 10, wherein the surfactant ispolysorbate
 80. 12. A pharmaceutical composition prepared byreconstituting the lyophilized composition of claim 11 in sterile water.13. A lyophilized composition produced by lyophilizing a pharmaceuticalcomposition comprising human acid glucosidase and a bulling agent. 14.The lyophilized composition of composition of claim 13 wherein thebulling agent is mannitol.
 15. The lyophilized composition of claim 14,further comprising a physiologically acceptable buffer.
 16. Thelyophilized composition of claim 15, wherein the buffer is a sodiumphosphate buffer.
 17. The lyophilized composition of claim 13, furthercomprising a surfactant.
 18. The lyophilized composition of claim 17,wherein the surfactant is polysorbate
 80. 19. A pharmaceuticalcomposition prepared by reconstituting the lyophilized composition ofclaim 13 in sterile water.